In this proposal, we will reveal mechanisms responsible for treatment resistance in patients with glioblastoma (WHO grade IV astrocytoma; GBM). Nearly all GBM patients experience tumor recurrence and treatment failure, limiting their median survival to a meager 15 months. Treatment resistance is often demonstrated by GBM recurrences that radiographically contact the subventricular zone (SVZ), the largest neural stem cell niche in the adult brain. Radiographic data in GBM patients suggest that GBMs display a tropism for the SVZ, and detectable SVZ contact by the tumor is a negative prognostic indicator. Data from mouse studies confirm this observation and reveal that CXCR4 receptor expressing (CXCR4+) GBM cells escape the tumor to migrate towards the SVZ in response to stromal-derived factor 1 (SDF1; CXCR4 ligand). Further, SVZ-derived SDF1 renders these cells more resistant to radiation therapy. Advancing from these observations, we will identify the intracellular molecular drivers of migration and radiation therapy resistance in CXCR4+ GBM cells from surgically resected human GBM samples. Activation of focal adhesion kinase (FAK) is an important signaling event downstream of SDF1/CXCR4. It is necessary in cell motility and alterations in its activity affect motility of glioma cell lines. Further, in other cancers, it plays an important role in repair of radiation induced DNA damage. We therefore hypothesize that FAK activation is necessary for CXCR4+ GBM cells to escape the tumor bulk and mediate resistance to radiation therapy. In human GBM surgical samples, we will detect and pharmacologically perturb FAK activation in the presence of SDF1 in vitro. Using high-dimensional mass cytometry, we will measure per-cell activation of CXCR4-responsive signaling pathways as well as modifications in cell migration and DNA repair proteins after radiation. We will generate xenografts with CXCR4+ GBM cells in mice and quantify GBM cells escaping from the tumor bulk with and without pharmacological inhibition of FAK. Finally, we will assess the impact on survival in mice of radiation therapy in combination with pharmacological inhibition of FAK. The results of this proposal will elucidate the role of FAK in mediating SVZ tropism and radiation therapy resistance of CXCR4+ GBM cells and test a novel, therapeutic approach for controlling GBM dissemination and increasing its sensitivity to radiation therapy.